Abstract 6688: Development of a liquid biopsy cfDNA assay for detection of multiple HPV types in cervical neoplasias

Abstract Background: Over 95% of cervical cancers and their precancerous lesions are caused by human papillomavirus (HPV). Molecular screening approaches for HPV detection are gaining promising data over cytologic analysis. Similarly, liquid biopsy-based approaches are becoming a powerful tool for cancer detection and monitoring. Materials and Methods: An HPV panel detecting 24 HPV types was developed suitable for liquid biopsy approaches. Here, HPV measurement was performed with an iPLEX multiplex PCR-based workflow combined with mass spectrometry-based analysis (MassARRAY System, Agena Bioscience). The assay was validated on HPV positive plasmid controls and cell lines. Furthermore, 52 cervical smear samples from HSIL positive women and 40 cfDNA samples (peripheral blood) from cervical cancer patients were screened using this approach. Results: According to already published ring trial results, the HPV types HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 58 and 59 were considered proficient with the required detection level of 50 and 5 international units (IU)/5 µl for HPV 16 and 18, and 500 and 50 HPV genome equivalents (GE)/5µl for the other HPV types (Arroyo Mühr et al., 2022). Further analyses showed a sensitivity of 0.03% for HPV16 and 0.33% HPV18 and 0.33% for the other tested HPV types. HPV DNA was detected in 94% of (49/52) smear samples analyzed. 86.5% were found positive for at least one of the high-risk HPV (hrHPV) types in the study cohort. Two of the three HPV negative samples were truly negative according to clinical data. In 15 samples, multiple HPV types were found, with positivity in up to five different HPV types. For smear samples a sensitivity of 98.14% and a positive predictive value of 0.98 (49/50) were thus obtained. The overall agreement between the MassARRAY and Seegene (from clinical records) HPV tests was 97.2% (κ=0.84). 27 of 40 (67.5%) cfDNA cervical cancer samples were tested positive for any HPV type. The distribution of HPV types showed most infections being due to hrHPV (25/27 plasma HPV positive). HPV positive samples were found in all FIGO stages, with 56.5% positivity in FIGO stage I and 87.5% in FIGO stage III and IV. Conclusions: The HPV assay showed reliable results for detecting a large number of HPV types in a multiplex mass spectrometry-based assay. A high sensitivity was achieved for both, cervical smear samples of HSIL patients as well as cfDNA from blood samples of cervical cancer patients. Citation Format: Johanna Herbst, Vanessa Vohl, Maroje Krajina, Katharina Prieske, Anna Jaeger, Markus Leffers, Leticia Oliveira-Ferrer, Yvonne Goy, Klaus Pantel, Alexander Sartori, Caren Vollmert, Linn Wölber, Katharina Effenberger, Harriet Wikman. Development of a liquid biopsy cfDNA assay for detection of multiple HPV types in cervical neoplasias [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6688.